11 In Vitro ValidationWe asked whether combined expression of Stat3 and C/EBPb in NSCs is sufficient to initiate mesenchymal gene expression and to trigger the mesenchymal properties that characterize high-grade gliomas. To do this, we used an early passage of the stable, clonal population of mouse NSCs known as C17.2 because its enhanced yet constitutively self-regulated expression of stemness genes permits its cells to be efficiently grown as undifferentiated monolayers in sufficiently large, homogeneous and viable quantities to ensure reproducible patterns of self-renewal and differentiation without ever behaving in a tumorigenic fashion in vitro or in vivo{Lee, 2007 #1332; Park, 2006 #1331; Parker, 2005 #1281}. Following ectopic expression of C/EBPb and a constitutively active form of Stat3 (Stat3C, Supplementary Fig. 1){Bromberg, 1999 #1312} in NSCs, we observed dramatic morphologic changes, consistent with loss of ability to differentiate along the neuronal lineage (Fig. 4a). Parental and vector-transfected NSCs have the classical spindle-shaped morphology that is associated with the neural stem/progenitor cell phenotype. When grown in the absence of mitogens, these cells display efficient neuronal differentiation characterized by extensive formation of a neuritic network (Fig. 4a, top-right panel). Conversely, expression of C/EBPb and Stat3C leads to cellular flattening and manifestation of a fibroblast-like morphology. Remarkably, depletion of mitogens resulted in additional flattening with complete loss of every neuronal trait

13 TCGA dataset on different networksPhillips (2)Sun (3)TCGA (1)221056841.9x10-71Cancer Genome Atlas Research Network, Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature Oct 23;455(7216):1061-82 Phillips, H.S., et al., Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell, (3): p3 Sun, L., et al., Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Cancer Cell, (4): p `Example of MaRINA results for inference of MR of the mesenchymal phenotype on the TCGA dataset by using three different interactomes: (1) TCGA, (2) Phillips, and (3) Sun. The Venn diagram shows a very significant overlap between the MR inferred from each interactome, showing that the three interactomes are functionally similar. The heatmap on the right shows the differential expression (first column) and the differential activity (NES, columns labelled as 1-3) inferred from each interactome for the MRs commonly identified on the three interactomes (top red and blue blocks), or identified only by two interactomes (bottom red and blue blocks).Distinct Programs with significant overlap across distinct datasets

22 Conclusions and ReflectionsCurrent emphasis on genes harboring genetic and epigenetic alterations may not be sufficientWe should also focus on Master Regulator and Master Integrator genesCurrent approach to biomarker discovery should be re-evaluated in a molecular interaction network context.It is not the genes/proteins that change the most but rather those that change most consistently. (mRNA is not informative)From GWAS (Genome-Wide Association Studies) to NBAS (Network-Based Association Studies)Califano A, Butte A, Friend S, Ideker T, and Schadt EE, Integrative Network-based Association Studies: Leveraging cell regulatory models in the post-GWAS era, Nat. Genetics, in press. Accessible in Nature Preceedings:One disease – One target – One drug Multi-target combinationsOptimal combination of drugs selected from a repertoire of safe, target-specific compounds using predictive tools.Identification of genetic dependencies (addictions) from Ex Vivo ModelsIdentification of candidate therapeutic agents from In Vitro mechanistic models.